Viral resistance to current antiretroviral medications and the side effects of these medications in patients necessitate the development of new antiretroviral agents that target additional stages of the HIV viral life cycle. Drugs that block the interaction between the HIV envelope protein gp120 and the coreceptor CCR5 are in development and will likely be available for the treatment of patients in the near future. As with other antiretroviral agents, viral resistance to these drugs is inevitable but little is known regarding the mechanisms of viral resistance and the consequences of resistance on clinical outcome, viral tropism, and disease pathogenesis. The specific aims of this proposal are to (1) clone and characterize HIV envelopes from patients on CCR5 inhibitors that have failed treatment, (2) perform structure-function studies of Env proteins to identify the molecular determinants associated with resistance, and (3) study the implications of resistance to CCR5 inhibitors on (a) susceptibility of HIV to other entry inhibitors and neutralizing antibodies and (b) alterations in host cell tropism and pathogenesis. The research design and methods for this project involve cloning Env genes from plasma samples from patients participating in CCR5 inhibitor trials. Samples from patients receiving aplaviroc have been donated by GlaxoSmithKline and samples from patients treated with miraviroc are being provided by Dr. Steve Deeks. Briefly, RNA is isolated from plasma, reverse transcribed into cDNA, and viral Env genes are amplified by PCR and cloned into expression vectors for cell-cell fusion and pseudovirus infectivity assays. Cloned Envs will be examined for sensitivity to CCR5 inhibitors, other fusion inhibitors, and neutralizing antibodies. Structure-function studies will be used to determine amino acid substitutions responsible for the development of resistance to R5 inhibitors. The consequences of these mutations will be assessed in viral fitness assays and alterations in tropism on cell lines and primary cells. The study of CCR5 inhibitors and the mechanisms by which HIV develops resistance to these compounds is directly relevant to clinical care of HIV-infected patients and may also indirectly aid in the pursuit of vaccines or immunotherapies by furthering our understanding of HIV entry.